Limits...
Rhabdomyosarcoma cells show an energy producing anabolic metabolic phenotype compared with primary myocytes.

Fan TW, Kucia M, Jankowski K, Higashi RM, Ratajczak J, Ratajczak MZ, Lane AN - Mol. Cancer (2008)

Bottom Line: Large differences were also evident in de novo biosynthesis of riboses in the free nucleotide pools, as well as entry of glucose carbon into the pyrimidine rings in the free nucleotide pool.The myocytes showed evidence of de novo synthesis of glycogen, which was absent in the Rh30 cells.The data further show that the mitochondria remain functional in Krebs' cycle activity and respiratory electron transfer that enables continued accelerated glycolysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, University of Louisville, KY, USA. twmfan@gmail.com

ABSTRACT

Background: The functional status of a cell is expressed in its metabolic activity. We have applied stable isotope tracing methods to determine the differences in metabolic pathways in proliferating Rhabdomysarcoma cells (Rh30) and human primary myocytes in culture. Uniformly 13C-labeled glucose was used as a source molecule to follow the incorporation of 13C into more than 40 marker metabolites using NMR and GC-MS. These include metabolites that report on the activity of glycolysis, Krebs' cycle, pentose phosphate pathway and pyrimidine biosynthesis.

Results: The Rh30 cells proliferated faster than the myocytes. Major differences in flux through glycolysis were evident from incorporation of label into secreted lactate, which accounts for a substantial fraction of the glucose carbon utilized by the cells. Krebs' cycle activity as determined by 13C isotopomer distributions in glutamate, aspartate, malate and pyrimidine rings was considerably higher in the cancer cells than in the primary myocytes. Large differences were also evident in de novo biosynthesis of riboses in the free nucleotide pools, as well as entry of glucose carbon into the pyrimidine rings in the free nucleotide pool. Specific labeling patterns in these metabolites show the increased importance of anaplerotic reactions in the cancer cells to maintain the high demand for anabolic and energy metabolism compared with the slower growing primary myocytes. Serum-stimulated Rh30 cells showed higher degrees of labeling than serum starved cells, but they retained their characteristic anabolic metabolism profile. The myocytes showed evidence of de novo synthesis of glycogen, which was absent in the Rh30 cells.

Conclusion: The specific 13C isotopomer patterns showed that the major difference between the transformed and the primary cells is the shift from energy and maintenance metabolism in the myocytes toward increased energy and anabolic metabolism for proliferation in the Rh30 cells. The data further show that the mitochondria remain functional in Krebs' cycle activity and respiratory electron transfer that enables continued accelerated glycolysis. This may be a common adaptive strategy in cancer cells.

Show MeSH

Related in: MedlinePlus

2-D 1H-13C HSQC analysis of Rh30 cells and myocytes. Both cells were cultured in [U-13C]-glucose for 24 hr before trichloroacetic acid extraction and HSQC measurement at 14.4 T. The spectra were recorded with 0.14 s acquisition in t2 and 34 ms in t1. The data table in t1 was linear predicted to 1024 complex points and zero filled to 2048, so that 13C-13C couplings (35–45 Hz) were resolved. Panel C displays the 2-D contour map of the Rh30 cell extract while panels B and A are respectively the 1-D 13C projection spectra of the 2-D contour maps for Rh30 and myocytes. Both A and B were normalized to cell dry weight but A was plotted at 10× scale relative to B.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2577687&req=5

Figure 7: 2-D 1H-13C HSQC analysis of Rh30 cells and myocytes. Both cells were cultured in [U-13C]-glucose for 24 hr before trichloroacetic acid extraction and HSQC measurement at 14.4 T. The spectra were recorded with 0.14 s acquisition in t2 and 34 ms in t1. The data table in t1 was linear predicted to 1024 complex points and zero filled to 2048, so that 13C-13C couplings (35–45 Hz) were resolved. Panel C displays the 2-D contour map of the Rh30 cell extract while panels B and A are respectively the 1-D 13C projection spectra of the 2-D contour maps for Rh30 and myocytes. Both A and B were normalized to cell dry weight but A was plotted at 10× scale relative to B.

Mentions: Figure 6 shows an expansion of the lactate region for the Rh30 cells shown in Fig 5. The 3-methyl resonances of both Ala and lactate (doublets at 1.53 and 1.4 ppm, respectively) display pairs of 13C satellite peaks displaced 126 Hz apart. These satellite peaks have a complex structure, which can be accounted for by considering additional couplings of these protons to 13C2 and 13C1 of the compounds (i.e. uniformly labeled) [35,47]. The presence of uniformly 13C-labeled lactate and Ala was also evident in the high-resolution 2-D 1H-13C HSQC or the 1-D 13C projection spectra of Rh30 and myocyte cell extracts, as illustrated in Figure 7. The respective doublet and triplet patterns of the β-(C3) and α-(C2) carbons of lactate and Ala (due to 13C-13C coupling) [36] is further consistent with the 1H TOCSY satellite patterns for these two metabolites (cf. Fig. 8). The NMR data were corroborated by the GC-MS analysis, where the m+3 (lactate+3) mass isotopomers predominated. These data together indicate that the three 13C in both lactate and Ala derived directly from the [U-13C]-glucose precursor via glycolysis.


Rhabdomyosarcoma cells show an energy producing anabolic metabolic phenotype compared with primary myocytes.

Fan TW, Kucia M, Jankowski K, Higashi RM, Ratajczak J, Ratajczak MZ, Lane AN - Mol. Cancer (2008)

2-D 1H-13C HSQC analysis of Rh30 cells and myocytes. Both cells were cultured in [U-13C]-glucose for 24 hr before trichloroacetic acid extraction and HSQC measurement at 14.4 T. The spectra were recorded with 0.14 s acquisition in t2 and 34 ms in t1. The data table in t1 was linear predicted to 1024 complex points and zero filled to 2048, so that 13C-13C couplings (35–45 Hz) were resolved. Panel C displays the 2-D contour map of the Rh30 cell extract while panels B and A are respectively the 1-D 13C projection spectra of the 2-D contour maps for Rh30 and myocytes. Both A and B were normalized to cell dry weight but A was plotted at 10× scale relative to B.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2577687&req=5

Figure 7: 2-D 1H-13C HSQC analysis of Rh30 cells and myocytes. Both cells were cultured in [U-13C]-glucose for 24 hr before trichloroacetic acid extraction and HSQC measurement at 14.4 T. The spectra were recorded with 0.14 s acquisition in t2 and 34 ms in t1. The data table in t1 was linear predicted to 1024 complex points and zero filled to 2048, so that 13C-13C couplings (35–45 Hz) were resolved. Panel C displays the 2-D contour map of the Rh30 cell extract while panels B and A are respectively the 1-D 13C projection spectra of the 2-D contour maps for Rh30 and myocytes. Both A and B were normalized to cell dry weight but A was plotted at 10× scale relative to B.
Mentions: Figure 6 shows an expansion of the lactate region for the Rh30 cells shown in Fig 5. The 3-methyl resonances of both Ala and lactate (doublets at 1.53 and 1.4 ppm, respectively) display pairs of 13C satellite peaks displaced 126 Hz apart. These satellite peaks have a complex structure, which can be accounted for by considering additional couplings of these protons to 13C2 and 13C1 of the compounds (i.e. uniformly labeled) [35,47]. The presence of uniformly 13C-labeled lactate and Ala was also evident in the high-resolution 2-D 1H-13C HSQC or the 1-D 13C projection spectra of Rh30 and myocyte cell extracts, as illustrated in Figure 7. The respective doublet and triplet patterns of the β-(C3) and α-(C2) carbons of lactate and Ala (due to 13C-13C coupling) [36] is further consistent with the 1H TOCSY satellite patterns for these two metabolites (cf. Fig. 8). The NMR data were corroborated by the GC-MS analysis, where the m+3 (lactate+3) mass isotopomers predominated. These data together indicate that the three 13C in both lactate and Ala derived directly from the [U-13C]-glucose precursor via glycolysis.

Bottom Line: Large differences were also evident in de novo biosynthesis of riboses in the free nucleotide pools, as well as entry of glucose carbon into the pyrimidine rings in the free nucleotide pool.The myocytes showed evidence of de novo synthesis of glycogen, which was absent in the Rh30 cells.The data further show that the mitochondria remain functional in Krebs' cycle activity and respiratory electron transfer that enables continued accelerated glycolysis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, University of Louisville, KY, USA. twmfan@gmail.com

ABSTRACT

Background: The functional status of a cell is expressed in its metabolic activity. We have applied stable isotope tracing methods to determine the differences in metabolic pathways in proliferating Rhabdomysarcoma cells (Rh30) and human primary myocytes in culture. Uniformly 13C-labeled glucose was used as a source molecule to follow the incorporation of 13C into more than 40 marker metabolites using NMR and GC-MS. These include metabolites that report on the activity of glycolysis, Krebs' cycle, pentose phosphate pathway and pyrimidine biosynthesis.

Results: The Rh30 cells proliferated faster than the myocytes. Major differences in flux through glycolysis were evident from incorporation of label into secreted lactate, which accounts for a substantial fraction of the glucose carbon utilized by the cells. Krebs' cycle activity as determined by 13C isotopomer distributions in glutamate, aspartate, malate and pyrimidine rings was considerably higher in the cancer cells than in the primary myocytes. Large differences were also evident in de novo biosynthesis of riboses in the free nucleotide pools, as well as entry of glucose carbon into the pyrimidine rings in the free nucleotide pool. Specific labeling patterns in these metabolites show the increased importance of anaplerotic reactions in the cancer cells to maintain the high demand for anabolic and energy metabolism compared with the slower growing primary myocytes. Serum-stimulated Rh30 cells showed higher degrees of labeling than serum starved cells, but they retained their characteristic anabolic metabolism profile. The myocytes showed evidence of de novo synthesis of glycogen, which was absent in the Rh30 cells.

Conclusion: The specific 13C isotopomer patterns showed that the major difference between the transformed and the primary cells is the shift from energy and maintenance metabolism in the myocytes toward increased energy and anabolic metabolism for proliferation in the Rh30 cells. The data further show that the mitochondria remain functional in Krebs' cycle activity and respiratory electron transfer that enables continued accelerated glycolysis. This may be a common adaptive strategy in cancer cells.

Show MeSH
Related in: MedlinePlus